J. Basic Microbiol. 30 (1990) 1,51-56

(“Boris KidriE” Institute of Chemistry, Ljubljana, Yugoslavia) Isolation and characterization of an alkaloid-blocked mutant of Claviceps paspali

ELIZABETAPERTOT, VLADIMIRA GABERC-POREKAR and HELENASOCIC

(Received 24 April I989/Accepted 3 May 1989) After UV-irradiation an alkaloid-blocked mutant of Claviceps pusgufi was isolated from an and amides (1 : 1) producing strain. The formation of ergometrine is blocked in this particular mutant strain which accumulates only lysergic acid amides. A significant morphological differentation of colonies of mutant as well as of mycelium in submerged cultures could be observed and total alkaloid yields are rather influenced.

The fungus Claviceps may be divided into numerous chemical races with different alkaloid spectra. Claviceps paspali strains in submerged cultures can produce : a-hydroxyethyl- amide of lysergic acid (ARCAMONEet al. 1961), ergometrine (GR~ERand TYLER1963) and A8*9-lysergicacid (KOBELet al. 1964), whereas several strains of C. purpurea produce mainly peptide alkaloids (GR~ERand ERGE 1970, SPALLA 1980, UDVARDY 1980, BIANCHIet a/. 1982). From the results of several investigators (KLEINEROVAand KYBAL 1969, KOBELet al. 1962, VOIGTand KEIPERT1967) it can be concluded that the composition of the alkaloid complex is genetically determined and that in nature biosynthetic pathway can proceed up to the peptide alkaloids, or be blocked more or less completely at some intermediate point. The spectra of saprophytically synthesized alkaloids may be changed either by mutation (STRNADOVA1964, 1967, KOBELand SANGLIER1973, GR~GER1979) or by changing the culture conditions (KRENet al. 1987, BREUELet af. 1982). The literature concerning the mutational biosynthesis in Claviceps is very sparse. An alkaloid- blocked mutant was isolated from an ergotoxine-producing C. purpurea strain, which accumulates -I and the corresponding chanoclavine-I aldehyde (MAIERet al. 1980). Another interesting mutant, obtained by multiple mutagenesis with UV light, was described by KRENet al. (1986). The block in the biosynthetic pathway of this mutant was only partial, since beside chanoclavine-I and chanoclavine-I aldehyde also small amounts of tetracyclic clavines and were produced. From a C. paspali lysergic acid a-hydroxyethylcarbinolamide producing strain a mutant capable of accumulating ergometrine was attained (MARNATIet al. 1975). In the present communication we describe another alkaloid-blocked mutant of C. paspali obtained after UV-irradiation. The mutant accumulates lysergic acid amides, while the formation of ergometrine is blocked.

Materials and methods

Microorganism : A Claviceps paspali (STEVENSand HALL)selected strain L-52, capable of producing simple lysergic acid derivatives, and from it derived the stable UV mutant strain designated as CP2, were employed in this study. Mutagenic treatment: A four-day old vegetative mycelium of C. paspali was homogenized in a WARiNG blendor for LO sec and mycelial fragments in 0.9% NaCl solution were exposed to UV irradiation (300 Jimz)

4’ 52 ELISABETAPERTOT et al, for time sufficient to kill 90-99% of the cells. Treated mycelial fragments were plate - cultured on potato- glucose agar. The resulting morphologically changed colonies were screened for alkaloid production and quali- tative changes of alkaloid spectrum. Culture media: Seed-stage medium was modified after ARCAMOMet al. (1961) and composed of 40 g/l mannitol, 10 g/l glucose, 10 g/l succinic acid, 3 g/l KH,PO,, 0,3 g/1 MgSO, . 7 H,O and 2 g/1 chick pea-meal in tap water; the pH of 5.2 was adjusted with ammonia before sterilization. Production-stage media: Three different media were used for the production cultures: Medium P-2: 150 g/l mannitol and 50 g/l peptone “Torlak” in distilled water; pH was about 6.8 without regulation (SOX er al. 1982). Medium MCK was modified after PERTOTet al. (1984) and composed of 50 g/l mannitol, 10 g/1 citric acid, 5 g/1 yeast extract “Torlak”, 1 g/1 KH,PO,, 0.3 g/1 MgSO, . 7 H,O and 20 g/l NaCl in tap water; the pH of 5.2 was adjusted with ammonia. Medium D (KOBELet al. 1964): 50 g/1 sorbitol. 36 g/1 succinic acid, 2 g/1 KH,PO,, 0.3 g/l MgSO, . 7 H,O, 1 mg FeSO, . 7 H,O and 10 mg/l ZnSO, 7 H,O in distilled water and was adjusted to a pH of 5.4 with NH,OH. All culture media were sterilized for 25 min at 121 “C, except medium D which was sterilized for 20 minutes at 108 “C. Culture conditions: C.paspali strains, both the parent and the mutant strain, were maintained on potato- glucose agar at 24 ’C and 14-21-day colonies were used for inoculating the seed medium. Screening experiments were carried out in two stages : seed-stage fermentation and production-stage fer- mentation. The 6-day old cultures were homogenized in a WARINGblendor for 10 sec and 10-15 % (V/V) were used to inoculate the production medium. The production-stage cultures were harvested after 15 days. The alkaloid content as well as the composition of the alkaloid complex were then determined. Submerged cultures were incubated on a rotary shaker (frequency 4 Hz, 50 mm stroke, 24 “C). Analytical methods: Mycelial dry weight: Culture broth (50 ml) was filtered, washed twice with water and dried at 85 “C to constant weight. Total alkaloids: Culture filtrate suitably diluted (2 ml) was mixed with VAN URKreagent (4 ml), which was prepared as described by AGURELL(1966), and the blue color determined spectrophotometrically with refe- rence to a standard solution of ergometrine (free base). The alkaloid composition was evaluated by thin-layer chromatography according to GROGERand ERGE (1963) and scanned on a CAMAGTLC scanner 11.

Results and discussion

The Claviceps paspali CP-2 mutant isolated after UV irradiation significantly differs from the original strain. Differences can be observed in morphological characteristics of colonies and differentiation of submerged mycelium as well as in the composition of the alkaloid spectrum. Colonies of the mutant, grown on potato-glucose agar have a smooth surface and white fluffy aerial mycelium, covered with yellow or greenish droplets of the exudate (“honeydew”), while colonies of the parent strain are compact with an even border, without fluffy aerial hyphae and without honeydew droplets (Fig. I). These morphological differences facilitated the selection. No conidia or arthrospores could be observed in the mutant’s honeydew droplets as it was noticed in some other C. puspali strains (RYLKOet a/. 1988). We have found that honeydew droplets contain polysaccharides as also reported for other Claviceps strains (RICKOVAet a/. 1986). Further we have found that polysaccharides are composed mainly of arabinose, galactose and xylose. The composition of sugars in the honeydew depends very probably on the composition of the medium used. In submerged cultures the mycelium of the mutant differentiated more or less into swollen, arthrosporoid-like cells and showed a tendency to fragmentize (Fig. 2). Such a morphological differentiation has never been observed with the wild strain of C. paspuli which retains filamentous structure throughout the fermentation. An alkaloid-blocked mutant of Cluviceps puspuli 53

Fig. 1 Colonies of C. paspali: parent strain (left) and mutant strain (right)

Fig. 2 Differentiated hyphae of a C. paspali mutant strain in submerged culture 54 ELIZABETAPERTOT et al.

Table 1 Percent content of individual alkaloids comprising the total alkaloid mixture of the parent and mutant strain

Alkaloid composition Media

Parent strain Blocked-mutant

P-2 D MCK P-2 D MCK

Lysergole 1.8 0.59 0.71 2.16 2.21 6.65 Lysergic acid amide 14.75 10.64 23.72 31.56 9.47 36.65 a-hydroxyethyl amide of lysergic acid 28.55 22.5 29.87 41.55 18.60 44.97 Ergometrine 32.5 I 2.82 24.59 - - - Ergometrinine 6.06 2.7 11.43 - - - Isolysergic acid amide 8.31 33.7 4.5 14.08 38.06 6.36 a-hydroxyethyl amide ' of isolysergic acid 5.22 26.29 3.45 10.32 27.71 5.09

Total alkaloids mg/I 2646 1028 1644 1552 1147 1257

Table 2 Biosynthetic activity of the parent strain and isolated mutant in submerged cultures in different media

Medium P-2 Medium D Medium MCK

Biomass Alkaloids Biomass Alkaloids Biomass Alkaloids g/l mgb s/l mg/l g/l mg/l

Parent strain 20.17 2647 9.63 1028 12.97 1644 Mutant strain 21.52 1552 14.43 1147 15.30 1257

Differences in the alkaloid composition between the CP-2 mutant and the parent strain are also significant and are presented in Table 1 where each value represents the arithmetic mean value of at least 12 individual cultures. The alkaloid composition varies somehow with the medium composition ; as can be seen from Table 1, in the P-2 and MCK media the 1-series simple derivatives of lysergic acid prevail while the medium D favours formation of d-forms, isolysergic acid derivatives, which are less interesting. As known, many compounds in the lysergic acid series exlubit pronounced pharmacological effects but the corresponding isolysergic acid derivatives are almost completely devoid of pharmacological activity. The main alkaloid components produced.'by the parent Claviceps paspali strain are the 1- and d-forms of ergometrine, lysergic acid a-hydroxyethylamide and lysergic acid amide. The CP-2 mutant synthesizes neither ergometrine nor ergometrinine in all three different nutrient media used, though the parent strain produced a significant quantity of both, particularly in the P-2 and MCK media. These results clearly indicate a block in the mutant's biosynthetic pathway. The amide components of ergometrine and lysergic acid cr-hydroxyethylamide are derived from L-alanine (GR~GERet al. 1968), the presumable intermediate being lysergylal- anine (REHACEK,1983). it seems that an enzyme responsible for reduction of lysergylalanine to ergometrine is blocked in the UV mutant, and only the oxydation step leading to the lysergic acid amides takes place (Fig. 3). An alkaloid-blocked mutant of Cluviceps puspuli 55

v3 CO-NH-q- H

CH3 ~H-COOti W-mutant "2 L-alanineLagH COOH ergometrine

\/ Y k H lysergic acid lysergylalanine

drhydroxyethyl amide of iysergic acid

Fig. 3 Biosynthetic pathway of simple lysergic acid derivatives. Bar indicates the position of the block

These results are just contrary to the modification of the biosynthetic pathway reported by MARNATIet al. (1975) who, by means of mutation with NTG, obtained a C. paspali strain capable of producing only ergometrine. Total alkaloid yields of the CP-2 mutant were found to be lower than yields exhibited by the original strain, irrespective of the medium used. On the contrary the mycelial dry weight of the mutant was always somewhat higher. The isolated mutant has proved to be perfectly stable. After several passages through agar and submerged cultures the morphological appearances and the composition of alkaloid spectra have remained fairly constant. In any case, the mutant described, accumulating only lysergic acid amides, is interesting not only from the research point of view but also as a useful tool for the semisynthetic preparation of lysergic acid and its other pharmacologically active derivatives.

Acknowledgement

The financial support of Research Community of Slovenia is gratefully acknowledged.

References

AGURELL.S., 1966. Biosynthesis of ergot alkaloids in Cluvicepspaspali. Acta Pharm. Suec., 3,ll- 12. ARCAMONE,F., CHAIN,E. B., FERRETTI,A,, MINGHETTI,A,, PENNELLA,P., TONOLO,A. and VERO,LIDIA, 1961. Production of a new lysergic acid derivative in submerged culture by a strain of Cluviceps puspuli STEVENSand HALL.Proc. Roy. SOC.,155 B, 25-54 56 ELIZABETAPERTOT et a/.

BIANCHI,M. L., CATTANEO,P. A,, CRESPI-PERELLINO,N., GUICCIARDI,A., MINGHETTI,A. and SPALLA,A., 1982. Mechanisms of control in the quantitative biosynthesis of lysergic acid alkaloids. In : Overproduction of Micribial Products (Eds: KRUMPHANZL,V., SIKYTA,B. and VANEK,Z.). Academis Press, London, 235 -242. BREUEL,K.. BRAUN,K., DAUTH,CH. and GR~GER,D., 1982. Qualitative changes in peptide alkaloid biosyn- thesis of by environmental conditions. Planta Medica, 44, 121- 122. GROGER,D., 1979. Saprophytische Gewinnung von Mutterkornalkaloiden. Pharmdzie, 34, 178. GROGER,D. and ERGE,D., 1963. Thin-layer chromatography of ergot alkaloids. Pharmazie, 18, 246-249. GROGER,D. and ERGE,D., 1970. Biosynthesis of the peptide alkaloids of Ctuvicepspurpureu. Z. Naturforsch. B, 25, 196- 199. GROGER,D., ERGE,D. und FLOSS,H. G., 1968. Uber die Herkunft der Seitenkette im D-Lysergsauremethyl- carbinolamid. Z. Naturforsch. 23b, 175-1 78. GROCER,D. and TYLER,V. E., 1963. Alkaloid production by Claviceps paspali in submerged culture. Lloydia, 26, 174-191. KLEINEROVA,E., KYBAL, J., 1969. Ergot alkaloids. 11. Spectrum ofergot alkaloids in the sclerotia of Claviceps purpurea and Claviceps paspali. Folia Microbiol., 14, 602-604. KOBEL,H., BRUNNER, R. and BRACK,A.. 1962. Comparison of alkaloid formation from Clavicepspurpurea in parasitic and saprophytic culture. Experientia, 18, 140- 141. KOBEL,H., SCHREIER,E. und RUTSCHMANN,J., 1964. 6-Methy1A8~9-ergolen-8-carbonsaure,ein news Ergo- lindenvat aus Kulturen eines Stammes von Claviceps paspali STEVENSand HALL.Helv. Chim. Acta, 47, 1052- 1064. . KOBEL,H. and SANGLIER,J. J., 1973. Qualitative changes in the alkaloid spectrum of Ciavicepspurpurea after mutation. In: Genetics of Industrial Microorganisms-Actinomycetes and Fungi (Eds. : Z. VANEK,Z. HOSTALEK,J. CUDLIN),Academia, Prague, 421 -425. KREN,V., PA~OUTOVA,S. SEDMERA, P., RYLKO,V. and REHACEK,Z., 1986. High-production mutant Claviceps purpurea 59 accumulating secoclavines. FEMS Microbiol. Lett., 37, 3 1-34. KREN,V., MEHTA.P.. RYLKO,V., FLIEGER,M., KOZOVA,J., SAJDL,P. and REHACEK,Z., 1987. Substrate regulation of elymoclavine formation by some saccharides. Zbl. Mikrobiol., 142. 71 -75. MAIERW., ERGE, D. and GROGER,D., 1980. Mutation biosynthesis in a strain Claviceps purpurea. Planta Medica, 40, 104- 108. MARNATI,M. P., MINGHETTI.A. and SPALLA,C., 1975. Genetic and metabolic relationship between alkaloid producing strains of Clavicepspaspali and Clavicepspurpurea. Conference on Medicinal Plants, Marianske Lazne, Abstracts p. 68. PERTOT,E., CADE~,J., MILICI~,S., and SOCIC.H.. 1984. The effect of citric acid concentration and pH on the submerged production of lysergic acid derivatives. Appl. Microbiol. Biotechnol., 20. 29-32. REHA~EK,Z., 1983. New trends in ergot alkaloid biosynthesis. Process Biochemistry, 22-30. RICICOVA,A,, POKORNY,V. and REHAEEK,Z., 1986. Characteristics of the saproohytic reference strain FA of Cluviceps paspali. Folia Microbiol., 31. 32-43. RVLKO,V., LINHARTOVA,R., SAJDL,P. and REHACEK.Z., 1988. Formation of conidia in a saprophytic strain Claviceps puspali producing simple lysergic acid derivatives. Folia Microbiol., 33. 425-429. SOCIC,H., PERTOT,E. and DIDEK-BRUMEC,M., 1982. The effect of media composition on the fermentative pro- duction of clavine ergot alkaloids. Vestn. Slov. Kem. Drus., 29, 323 -329. SPALLA,C., 1980. Production of ergot alkaloids by fermentation. In : Iiidole and Bioenergetically Related Alkaloids (Eds.: J. D. PHILIPSONand M. H. ZENK).Academic Press, London. New York, 271-2284, STRNADOVA,K., 1964. Ultraviolet mutant in Chireps purpurea. Planta Medica, 12, 521 -527. STRNADOVA,K., 1967. Alkaloid content and type of auxotrophic UV-mutants of ergot (Claviceps purpurea) in parasitic culture of rye. Flora Abt. A, 157, 517-523. UDVARDY-NAGY,E., 1980. Consideration of the development of an ergot alkaloid fermentation process. Process Biochemistry, 15. 5-8. VOIGT,R. and KEIPERT,S., 1967. Modification of the alkaloid spectrum in Clavicepspurpurea in saprophytic culture. 11. Effect of the culture medium. Pharmazie, 22, 329-336.

Mailing address: ELIZABETAPERTOT, Boris KidriE Institute of Chemistry, Hajdrihova 19. 61 115 Ljubljana, Yugoslavia